Protection Of Pads Inhibitor In Rats With Hemorrhagic Shock Induced Acute Lung Injury

Part ?Investigation of neutrophil Cit H3 expressions in hemorrhage shock rat modelsObjective To investigate the effect of hemorrhagic shock(HS)on Cit H3 expressions in both circulating and pulmonary neutrophil via an in vivo rat model.Materials and Methods HS model was established by femoral artery bleeding for 10 min,up till 40%of total circulation volume,using male Sprague-Dawley rat.The circulating neutrophils were isolated.Western Blot was performed to measure the expression of total H3 and Cit H3 in circulating neutrophils.Immunohistochemistry was used to detect Cit H3 expression in lung tissues.Results The expression of Cit H3 in HS group was significantly higher than that in sham operation group(P<0.05),while no significant difference of total H3 expression was observed between the two groups.Pulmonary immunohistochemistry data showed that CitH3 mainly localized in neutrophil nuclear and the expression of CitH3 in HS group was significantly higher than that in the Sham group.Conclusion Rats HS significantly increased CitH3 expression in both circulating and lung neutrophils.Part ? Effects of PAD4 inhibitor on acute lung injury and survival rate following hemorrhage shock in ratsObjective To investigate the protective effect of a novel PAD inhibitor,YW3-56,in hemorrhagic shock induced acute lung injury.Materials and Methods HS model was established by femoral artery bleeding by using male Sprague-Dawley rat.In the survival experiment,anesthetized male Wistar-Kyoto rats(n=10/group)were subjected to 55%blood loss,treated with or without YW3-56(10mg/kg,intraperitoneally).Survival was monitored for 12 h.In the non-survival experiment,morphorlogic changes of the lungs were examined.Levels of circulating cytokine-induced neutrophil chemoattractant 1(CINC-1)and myeloperoxidase(MPO)in the lungs were measured by ELISA.Expression of lung intercellular adhesion molecules-1(ICAM-1)was also determined by Western blotting.Results YW3-56 treatment improved survival rate from 20%to 60%in lethal HS rat model.Compared to the sham groups,pulmonary MPO activity and ICAM-1 expression in the HS group were significantly increased,and acute lung injury was associated with a higher degree of CINC-1 levels in serum.Intra-peritoneal delivery of YW3-56 significantly reduced pulmonary MPO and ICAM-1 expression and attenuated acute lung injury.Conclusion YW3-56 significantly improved survival rate and decreased lung inflammation in HS rats.YW3-56 significantly inhibited the CINC-1 and ICAM-1 expressions and MPO activity in lung tissues of HS rats.YW3-56 may become one important strategy for the treatment of HS.Part ? Effects of PAD4 inhibitor on expression of Cit H3 and NETs formationObjective To investigate the effects of YW3-56,a Peptidylarginine deiminases(PADs)inhibitor,on Cit H3 expression and pulmonary neutrophil extracellular networks(NETs)formation in both in vivo and in vitro models.Materials and Methods HS model was established by femoral artery bleeding by using male Sprague-Dawley rat.The circulating neutrophils were isolated from the animal model either treated or non-treated with YW3-56.On the other hand,HL-60 cells were induced to neutrophil like cells and then treated with LPS,calcium ionophore(Cal+)or hypoxia/reoxygenation(H/R).Western blot was employed to detect the levels of Cit H3,total H3,Cit H4,total H4 expression in neutral granulocyte.The expression of Cit H3 in lung tissue was determined with immunofluorescence.MPO and NETs was analyzed by immunofluorescence.Results The increased expression of Cit H3 and the formation of NETs have been observed in HS animal model,while increased the expression of Cit H3 and the NETs formation were significantly reduced after the intervention of YW3-56.Neutrophil like cells treated with LPS,calcium ionophore(Cal+)or hypoxia/reoxygenation(H/R)showed a significant increase of Cit H3 and Cit H4 expression and NETs formation.Pretreatment with YW3-56(5.0 uM)significantly inhibited Cit H3 and Cit H4 expression,NETs formation and MPO expression.Conclusion PADs inhibitor YW3-56 significantly reduced Cit H3 expression and the formation of NETs in HS rats.YW3-56 down-regulate the Cit H3 expression,NETs formation and MPO expression in H/R treated neutrophil like cells.YW3-56 may be used as one important treatment strategy for HS.Part ? Effects of NETs on the secretion of proinflammatory cytokines and chemokines in macrophagesObjective To analyze the effects of NETs on proinflammatory cytokines and chemokines secretion by macrophagesMaterials and Methods The HL-60 cell line was cultured and induced differentiation by ATAR in vitro.Calcium ionophore(Cal+)were used to stimulate the cells under the pretreatment of YW3-56 or placebo.The NETs were extracted from liquid mixture and were applied to stimulate macrophages cell line RAW 264.7.The supernatant tumor necrosis factor-a(TNF-a),interleukin-6(IL-6),cytokine-induced neutrophil chemoattractant-1(CINC-1)levels were measured using ELISA.Results NETs extracted from YW3-56 pretreatment samples showed significant lower effect on stimulating macrophages to produce inflammatory cytokines TNF-a,IL-6 and CINC-1,as compared to untreated NETs extrats.Administration of anti-Cit-H3,Cit-H4 antibody,and DNase I to RAW 264.7 cells also significantly decreased macrophages TNF-a and CINC-1 expressions after NETs stimulation.Conclusion YW3-56 pretreatment inhibited the formation NETs,subsequently inhibited macrophage to secret proinflammatory factors.Neutralization of NETs component Cit H3 or Cit H4 can also reduce the proinflammatory response of macrophages.NETs may play an important role in the activation of acute lung injury induced by HS.